Generation of Th1 and Th17 Cells from 2D2 Mice

Spleens were collected from 2D2 TCR transgenic mice, and a single-cell suspension was prepared and cultured as previously described (17 (link)). Briefly, splenocytes were cultured in the presence of 20 µg/ml MOG_35-55 peptide (GenScript Corporation). Th1 polarization was induced by in vitro stimulation with 1 ng/ml IL-12 (R&D Systems) and 10 µg/ml anti-IL-4 antibody (clone 11B11, hybridoma kindly provided by E. C. Butcher, Stanford University), while Th17 polarization was induced by using 5 ng/ml TGF-β, 20 ng/ml IL-6 and 2 ng/ml IL-23 (all from Miltenyi Biotech or R&D Systems), as well as 10 µg/ml anti-IL-4 antibody (as above) and 10 µg/m anti-IFNγ antibody (clone HB170, R&D Systems). After 4 days in culture, Th1 and Th17 cells were supplemented with IL-2 (20 U/ml) or IL-7 (10 ng/ml) respectively for other 72 h. MOG_35-55-specific Th1 and Th17 cells were then isolated using a Ficoll-Paque density gradient (GE Healthcare Life Sciences) and re-stimulated for 3 days in the presence of irradiated splenocytes as APCs (APC:T cell ratio = 5:1) with the same protocol used for the first stimulation. Th1 and Th17 cells were then isolated as described above and supplemented for one further day with IL-2 or IL-7, respectively. Th1 and Th17 polarization was confirmed by flow cytometry (Supplementary Figures 1A, B) as previously described (17 (link)).